This study investigated the effect of scallop plasmalogen (SP) on intestinal barrier function and the potential role of the gut microbiota in this effect. C57BL/6J male mice were divided into four groups, which were fed respectively with a low-fat diet (LFD), a high-fat diet (HFD), LFD + SP, and LFD + SP. Immunohistochemical staining and 16S rRNA sequencing were used to evaluate the effect of SP on growth index, serum inflammatory factor levels, intestinal permeability, intestinal barrier function and gut microbiota. The results showed that SP reduced the liver index and lowered serum proinflammatory factor levels in HFD-fed mice. SP also improved intestinal permeability, as evident by a decrease in the serum level of lipolyaccharide and a reduction in the circulating level of fluorescein isothiocyanate (FITC)-dextran in HFDfed mice. Meanwhile, SP maintained the structural morphology of the ileum and increased the ratio of villus height to crypt depth in HFD-fed mice. This may be attributed to the protective effect of SP on intestinal barrier function. SP alleviated intestinal mucosal barrier damage, restored the number of ileal goblet cells, protected the integrity of the intestinal epithelial barrier, and reversed the decrease in zonula occludens-1 (ZO-1) and occludin expression. Furthermore, SP significantly regulated the intestinal microbial composition, promoting the proliferation of beneficial bacteria related to intestinal barrier function, such as Colidextribacter and Lachnospiraceae_NK4A136_group. In summary, SP has protective effect against intestinal barrier damage and gut microbiota dysbiosis, and thus maintains intestinal health.